THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 23, pp.
19315–19325, June 1, 2012
Purification and Identification of Naringenin
7-O-Methyltransferase, a Key Enzyme in Biosynthesis
of Flavonoid Phytoalexin Sakuranetin in Rice*□
Received for publication, February 9, 2012, and in revised form, April 3, 2012 Published, JBC Papers in Press, April 9, 2012, DOI 10.1074/jbc.M112.351270
Takafumi Shimizu‡, Fengqiu Lin‡, Morifumi Hasegawa§, Kazunori Okada‡1, Hideaki Nojiri‡, and Hisakazu Yamane‡2
From the ‡Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo and the §College of Agriculture,
Ibaraki University, 3-21-1 Chuo, Ami, Ibaraki, Japan
Background: Sakuranetin is a major rice phytoalexin and a potential pharmaceutical agent. Rice naringenin 7-O-methyl-
transferase (OsNOMT), the key enzyme for sakuranetin biosynthesis, was previously unknown.
Results: We isolated OsNOMT and identified Os12g0240900 as OsNOMT.
Conclusion: Stress-induced OsNOMT regulates sakuranetin biosynthesis in rice.
Significance: Identification of OsNOMT enables the production of large amounts of sakuranetin through transgenic rice and
microorganisms.
Sakuranetin, the major flavonoid phytoalexin in rice, is
induced by ultraviolet (UV) irradiation, CuCl2 treatment, jas-
monic acid treatment, and infection by phytopathogens. It was
recently demonstrated that sakuranetin has anti-inflammatory
activity, anti-mutagenic activity, anti-pathogenic activities
against Helicobacter pylori, Leishmania, and Trypanosoma and
contributes to the maintenance of glucose homeostasis in
animals. Thus, sakuranetin is a useful compound as a plant anti-
biotic and a potential pharmaceutical agent. Sakuranetin is
biosynthesized from naringenin by naringenin 7-O-methyl-
transferase (NOMT). In previous research, rice NOMT
(OsNOMT) was purified to apparent homogeneity from UV-
treated wild-type rice leaves, but the purified protein, named
OsCOMT1, exhibited caffeic acid O-methyltransferase (COMT)
activity and not NOMT activity. In this study, we found that
OsCOMT1 does not contribute to sakuranetin production in rice
in vivo, and we purified OsNOMT using the oscomt1 mutant. A
crude protein preparation from UV-treated oscomt1 leaves was
subjected to three sequential purification steps, resulting in a 400-
fold purification from the crude enzyme preparation. Using SDS-
PAGE, the purest enzyme preparation showed a minor band at an
apparent molecular mass of 40 kDa. Two O-methyltransferase-like
proteins, encoded by Os04g0175900 and Os12g0240900, were
identified from the 40-kDa band by MALDI-TOF/TOF analysis.
Recombinant Os12g0240900 protein showed NOMT activity, but
the recombinant Os04g0175900 protein did not. Os12g0240900
expression was induced by jasmonic acid treatment in rice leaves
prior to sakuranetin accumulation, and the Os12g0240900 protein
showed reasonable kinetic properties to OsNOMT. On the basis of
these results, we conclude that Os12g0240900 encodes an
OsNOMT.
* This work was supported by the Program for Promotion of Basic Research
Activities for Innovative Biosciences.
□
S
This article contains supplemental Figs. S1–S4 and Table S1.
The nucleotide sequence(s) reported in this paper has been submitted to the
DDBJ/GenBankTM/EBI Data Bank with accession number(s) AB692949.
1
To whom correspondence should be addressed. Tel.: 81-3-5841-3070; Fax:
81-3-5841-3070; E-mail: ukokada@mail.ecc.u-tokyo.ac.jp.
2
Present address: Dept. of Biosciences, Teikyo University, Toyosatodai,
Utsunomiya, Tochigi, Japan.
JUNE 1, 2012 • VOLUME 287 • NUMBER 23
© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
S
When plants are attacked by pathogenic microorganisms,
they respond with a variety of defense reactions, including the
production of secondary metabolites called phytoalexins (1),
which serve as plant antibiotics. In rice, 15 phytoalexins have
been isolated and characterized, including 14 diterpenes,
namely momilactones A and B, phytocassanes A–E, oryzalexins
A–F, and oryzalexin S (2–10), and one flavonoid phytoalexin,
sakuranetin (Fig. 1) (11). Among them, sakuranetin is consid-
ered to be one of the most biologically important phytoalexins
in terms of its high antimicrobial activity and high accumula-
tion in rice leaves infected by Magnaporthe oryzae, one of the
major photopathogenic fungi (11). In addition, it was recently
reported that sakuranetin has anti-inflammatory activity by
inhibiting 5-lipoxygenase, which is involved in arachidonic acid
metabolism in animal cells (12), anti-mutagenic activity (13),
anti-Helicobacter pylori activity by inhibiting ␤-hydroxyacyl-
acyl carrier protein dehydratase (14), and antileishmanial and
antitrypanosomal activities (15), and that sakuranetin can
induce adipogenesis of 3T3-L1 cells through enhanced expres-
sion of peroxisome proliferator-activated receptor ␥2 to con-
tribute to the maintenance of glucose homeostasis in animals
(16). Thus, sakuranetin is a useful compound as a plant antibi-
otic and a potential pharmaceutical agent.
Sakuranetin is biosynthesized from naringenin by S-adenosyl-
3
L-methionine (AdoMet) -dependent naringenin 7-O-methyl-
transferase (NOMT) (Fig. 1) (17). Naringenin is the first flavonoid
transformed from naringenin chalcone by chalcone isomerase and
is also a key biosynthetic intermediate to an isoflavone and a vari-
ety of flavones. Therefore, NOMT plays a key role in sakuranetin
biosynthesis at a branching point from a common flavonoid bio-
synthetic pathway.
Sakuranetin was first identified from the cortex of the bark of
a cherry tree (Prunus spp.) as an aglycone of sakuranin (18) and
3
The abbreviations used are: AdoMet, S-adenosyl-L-methionine; NOMT, nar-
ingenin 7-O-methyltransferase; COMT, caffeic acid O-methyltransferase;
OMT, O-methyltransferase; JA, jasmonic acid; RACE, rapid amplification
of cDNA ends; qRT, quantitative RT; Rubisco, ribulose-bisphosphate
carboxylase/oxygenase.
JOURNAL OF BIOLOGICAL CHEMISTRY 19315
Purification and Identification of Rice Sakuranetin Synthase
FIGURE 1. Biosynthesis of sakuranetin from naringenin.
then found in rice and several other plant species, including
Artemisia campestris, Prunus spp., Baccharis spp., Betula spp.,
and Juglans spp. (19). However, NOMT has not been identified
from such plant species. Although an O-methyltransferase
(OMT) from barley (F1-OMT) that mainly methylates apigenin to
form genkwanin has weak NOMT activity, sakuranetin has not
been identified in barley (20). It was also reported that another
OMT isolated from Streptomyces avermitilis (SaOMT-2) is able to
catalyze the NOMT reaction, but SaOMT-2 has broad substrate
specificity against isoflavones, flavones, and a flavanone, and its
biological function remains unknown (21).
Although sakuranetin is not found in healthy rice leaves, its
biosynthesis is rapidly induced by both biotic and abiotic
stresses, such as infection with phytopathogens such as M.
oryzae, Xanthomonas oryzae, and Ditylenchus angustus, infesta-
tion with Sogatella furcifera, ultraviolet (UV) irradiation, and treat-
ment with CuCl2 or jasmonic acid (JA) (11, 17, 22–25). Previously,
it was reported that NOMT was purified to apparent homogeneity
(⬃985-fold) from crude extracts of UV-treated wild-type rice
leaves (22). However, the amino acid sequence of the purified
protein was highly homologous to that of a caffeic acid 3-O-
methyltransferase (COMT) from maize. In fact, the recombi-
nant protein expressed in Escherichia coli showed COMT but
not NOMT activity (26), and this enzyme was named
OsCOMT1. These results suggest at least two possibilities as
follows: one is that OsCOMT1 is a major component and the
rice NOMT (OsNOMT) is a minor one in the purified fraction.
If this is the case, isolation of OsNOMT might be quite difficult
because of the masking effect by OsCOMT1. Another is that
OsCOMT1 is involved in NOMT enzymatic activity in rice in
vivo but that a post-translational modification and/or the pres-
ence of an interacting factor is needed to show NOMT activity.
In the former case, if UV-irradiated leaves of the OsCOMT1
tDNA insertion mutant oscomt1 are used as plant material,
OsNOMT is expected to be purified quite efficiently without
masking by OsCOMT1. In the latter case, NOMT activity will
not be induced in oscomt1 mutant leaves even after elicitation.
In this study, we first confirmed that NOMT activity is
induced by elicitor treatment in the oscomt1 mutant leaves. We
then purified and identified OsNOMT from UV-irradiated
leaves of the oscomt1 mutant. Kinetic analysis of recombinant
OsNOMT and expression analysis of the OsNOMT gene in rice
plants were also performed. This is the first report on the clon-
19316 JOURNAL OF BIOLOGICAL CHEMISTRY
ing and characterization of OsNOMT as a sakuranetin synthase
in rice.
EXPERIMENTAL PROCEDURES
Chemicals—Racemic naringenin, luteolin, caffeic acid, and
ferulic acid were purchased from Kanto Chemical, Co., Inc.
(Tokyo, Japan). Racemic daidzein, biochanin A, and sinapic
acid were purchased from Sigma. Apigenin, kaempferol, myric-
etin, and quercetin were purchased from Wako Pure Chemical
Industries (Osaka, Japan). Racemic liquiritigenin was pur-
chased from Funakoshi Co., Ltd. (Tokyo, Japan). All chemicals
used in matrix-assisted laser desorption/ionization two-stage
time-of-flight mass spectrometry (MALDI-TOF/TOF) ana-
lyses were of analytical grade. In addition, 4-sulfophenyl iso-
thiocyanate, ␣-cyano-4-hydroxycinnamic acid, sodium bicar-
bonate, and ammonium bicarbonate were purchased from
Sigma.
Plant Materials, Growth Conditions, and Elicitation—The
oscomt1 mutant (PFG-2B-50240, Oryza sativa L. cv. Dongjin)
was searched for in the Rice Functional Genomic Express Data-
base as a tDNA insertion mutant of Os08g0157500 (TIGR ID,
LOC_Os08g06100) and purchased from Postech Biotech Cen-
ter. Because the seeds of the oscomt1 mutant we obtained were
from the F1 generation, the F1 generation plants were analyzed
by genomic PCR using specific primers for OsCOMT1 and
tDNA to separate oscomt1 homozygotes, heterozygotes,
and wild-type rice plants, and the F2 generation seeds were
obtained from the respective plants. We used these F2 genera-
tion seeds of homozygotes and wild type as “oscomt1” and “wild
type” in the experiments on the characterization of the oscomt1
mutant and purification of OsNOMT. Primers used for
genomic PCR are described in Fig. 2A and supplemental Table
S1. Wild-type rice (O. sativa L. cv. Nipponbare) plants were
used for 5⬘- and 3⬘-RACE of OsNOMT mRNA and for analysis
of the effects of JA treatment or inoculation of M. oryzae spores
on the expression of OsNOMT and quantification of accumu-
lated sakuranetin.
To grow rice plants, seeds were sterilized with 2.5% sodium
hypochlorite solution (Kanto Chemical) for 30 min and then
washed with sterilized water. Surface-sterilized seeds were
incubated on 0.7% agar gel containing 0.1% of the liquid fertil-
izer Hanakoujou (Sumika-Takeda Garden Products, Tokyo,
Japan) for 10 days at 28 °C in the light. Germinated seeds were
VOLUME 287 • NUMBER 23 • JUNE 1, 2012
 Purification and Identification of Rice Sakuranetin Synthase

FIGURE 2. Genome structure and gene expression of OsCOMT1 in the oscomt1 mutant. A, tDNA sequence is inserted 374 bp downstream of the transcrip-
tion initiation site of OsCOMT1 in the oscomt1 mutant. Black arrowheads (#1, #2, and #3) indicate the locations of primers used for genomic PCR, and white
arrowheads indicate the locations of primers used for expression analysis. B, results of genomic PCR of the oscomt1 mutant allele. C, transcriptional expression
of OsCOMT1 in wild-type rice and the oscomt1 mutant. Ubiquitin expression was used as a control.

then transplanted into a mixture of vermiculite and artificial com-
post, Bonsol (Sumitomo Chemical, Tokyo, Japan), to grow in a
greenhouse (12 h of light at 28 °C/12 h of dark at 25 °C). Three-
month-old plants were used for subsequent experiments.
For UV irradiation, excised rice leaves were floated on dis-
tilled water and incubated for 20 min at 20 cm under a UV lamp
in a biological safety cabinet. Then the irradiated leaves were
incubated at 28 °C under continuous white light conditions. For
JA or CuCl2 treatment, a leaf disk (6 mm in diameter) from the
uppermost leaf blades of rice plants was floated on 100 ␮l of an
assay solution (500 ␮M of JA or CuCl2) in each well of 96-well
plastic plates.
Expression Analysis—RT-PCR and qRT-PCR were used to
determine the level of gene expression. Total RNA extracted
from plant tissue using Sepasol-RNA I Super (Nacalai Tesque,
Inc., Tokyo, Japan) was used to synthesize cDNA by RT reac-
tion using a Quantitect RT kit (Qiagen K.K., Tokyo, Japan).
With the cDNA, RT-PCR and qRT-PCR were performed to
determine the level of gene expression. For qRT-PCR, SYBR
Green technology on an ABI PRISM 7300 real time PCR system
(Applied Biosystems) was used. Raw data from qRT-PCR were
analyzed using the standard curve method, and the results were
expressed as relative mRNA values normalized to the expression
level of ubiquitin (OsUBQ). Primers used for expression analysis
are described in supplemental Table S1.
Extraction and Quantification of Naringenin and Sakurane-
tin from Rice Leaves—Plant tissue (40 –200 mg fresh weight per
sample) was homogenized by Multi Beads Shocker (Yasui Kikai,
Osaka, Japan) and suspended in 2 ml of phytoalexin extraction
solvent (ethanol/water/acetonitrile/acetic acid, 79:13.9:7:0.1,
v/v). The sample was centrifuged at 8,000 ⫻ g for 15 min at 4 °C.
The supernatant was collected to a vial and subjected to deter-
mination of phytoalexins by liquid chromatography two-stage
mass spectrometry (LC-MS/MS), which was composed of API-
3000 with an electrospray ion source (AB SCIEX) and an Agi-
lent 1100 HPLC instrument (Agilent Technologies) equipped
with a PEGASIL ODS SP100 column (150 mm long, 2.1 mm in
diameter; Senshu Scientific, Tokyo, Japan). The analytical con-
ditions were the same as the method described by Shimizu et al.
(27). Naringenin and sakuranetin levels were determined with
combinations of the precursor and product ions of m/z 273/153
for naringenin and m/z 287/167 for sakuranetin in the multiple
reaction monitoring mode. The retention times of naringenin
and sakuranetin were 2.5 and 3.3 min, respectively.
Preparation of Crude Protein Extract from Rice Leaves—All
steps were carried out at 4 °C unless otherwise stated. Rice
leaves were homogenized by using Polytron (Kinematica AG,
Lucerne, Switzerland) with a 3-fold (v/w) volume of buffer A
(0.2 M Tris-HCl (pH 8.5), containing 10% (v/v) glycerol). The
homogenate was filtered through four layers of Miracloth (Cal-
biochem), and the filtrate was centrifuged at 10,000 ⫻ g for 30
min, with the resulting supernatant used as a crude protein
extract.
NOMT and COMT Enzymatic Activity Assays—The stan-
dard assay mixture consisted of enzyme preparation (⬃500 ng
of protein), 300 ␮M racemic naringenin or caffeic acid, 300 ␮M
AdoMet, and 0.1 M Tris-HCl (pH 8.5) (containing 5 mM DTT
and 1 mM EDTA) in a final volume of 50 ␮l. After an 18-h
incubation at 28 °C, the reaction was terminated by adding 5 ␮l
of 1 M HCl. The resulting products were extracted three times
with 60 ␮l of ethyl acetate, evaporated to dryness, and finally
dissolved in 100 ␮l of the phytoalexin extraction solvent. The
content of sakuranetin or ferulic acid was quantified using an
API-3000 LC-MS/MS system. Ferulic acid levels were deter-
mined with combinations of m/z 195/177 in the multiple reac-
tion monitoring mode.
Purification of OsNOMT—All steps were carried out at 4 °C
unless otherwise stated. A crude protein extract (620 ml) from
200 g, fresh weight, of oscomt1 mutant rice plants 48 h after UV
irradiation was applied to the sequential three-step purification
described below.
Ammonium Sulfate Precipitation—Well ground ammonium
sulfate powder (108.7 g; Kanto Chemical) was added to the
crude protein extract, and the mixture was gently agitated for
2 h. The mixture was then centrifuged at 10,000 ⫻ g for 30 min.
The supernatant was collected to a clean beaker; 77.5 g of
ammonium sulfate powder was added, and the mixture was
gently agitated for 2 h. After that, the mixture was centrifuged

JUNE 1, 2012 • VOLUME 287 • NUMBER 23 JOURNAL OF BIOLOGICAL CHEMISTRY 19317

Purification and Identification of Rice Sakuranetin Synthase
at 10,000 ⫻ g for 30 min. The supernatant was removed, and 40
ml of buffer A was added to the pellet. Next, 48 ml of the
obtained protein solution was ultrafiltered (10-kDa cutoff) and
finally resolved in 12 ml of buffer A, and the resultant solution
was subjected to the subsequent purification.
Anion Exchange on DEAE Column—The protein solution (10
ml) was loaded to an AKTA fast protein LC (FPLC) system (GE
Healthcare) equipped with a pre-equilibrated Hi PrepTM 16/10
DEAE FF column (GE Healthcare). Buffer A was used as a pre-
equilibration buffer. The applied proteins were washed with 50
ml of buffer A at a flow rate of 2 ml/min. After that, a 50-min
linear gradient (0 – 60% buffer B (buffer A containing 1 M KCl))
was applied as an elution step with a flow rate of 2 ml/min and
then the column was washed with 100 ml of 100% buffer B. The
eluate was collected in steps of 5 ml each, checked for NOMT
enzymatic activity, and the active fractions were pooled. The
pooled samples were mixed together, ultrafiltered (10-kDa cut-
off), and finally resolved in 6 ml of buffer A, with the product
used for subsequent purification.
Affinity Chromatography—Affinity chromatography on aden-
osine-agarose was performed as described previously (22).
First, 5⬘-AMP-agarose (5 ml; Sigma) was washed with distilled
water and incubated with 800 units of calf intestinal alkaline
phosphatase (Sigma) in calf intestinal alkaline phosphatase
buffer (pH 9.0) in a total volume of 10 ml. The gel was then
dephosphorylated for 24 h at 37 °C by gentle agitation, trans-
ferred to an open column (15 mm inner diameter), and washed
with 50 ml of distilled water and then with 50 ml of buffer B.
Finally, the column was equilibrated with 100 ml of buffer A.
The DEAE-purified protein solution (1 ml, ⬃18.7 mg of pro-
tein) was directly applied onto the adenosine-agarose gel col-
umn. The column was washed first with 50 ml of buffer A and
then with 50 ml of buffer B with gravity flow. Elution was done
with 25 ml of 4 mM AdoMet in buffer B. Fractions (5 ml each)
were collected, and the NOMT enzymatic activity of each frac-
tion was checked. Active fractions were combined, ultrafiltered
(10-kDa cutoff), and finally dissolved in 1.2 ml of buffer A. The
resultant solution was subjected to SDS-PAGE.
In-gel Protein Digestion—Protein bands on SDS-polyacryl-
amide gels were enzymatically digested in-gel as described pre-
viously (28) using modified porcine trypsin (Promega). The
resultant gel pieces were washed with 50% acetonitrile and vac-
uum dried, then rehydrated with trypsin solution (8 –10 ng/␮l
50 mM ammonium bicarbonate (pH 8.7)), and incubated for
8 –10 h at 37 °C.
Identification of Purified Protein by MALDI-TOF and
MALDI-TOF/TOF Analysis—For identification of components
of the 40-kDa band in SDS-PAGE (Fig. 4A) by peptide mass
fingerprinting with MALDI-TOF/TOF, in-gel digested protein
samples were analyzed using the Applied Biosystems 4700 pro-
teomics analyzer with TOF/TOFTM ion optics. Both MS and
MS/MS data were acquired with a Nd:YAG laser with a 200 Hz
repetition rate, and up to 4,000 shots were accumulated for
each spectrum. The MS/MS mode was operated with 1 keV
collision energy; air was used as the collision gas such that nom-
inally single collision conditions were achieved. Both MS and
MS/MS data were acquired using the instrument default cali-
bration, without applying internal or external calibration.
19318 JOURNAL OF BIOLOGICAL CHEMISTRY
Sequence tag searches were performed with the program
MASCOT.
For identification of components of the 45- and 52-kDa
bands in SDS-PAGE (Fig. 4A) by peptide mass fingerprinting
with MALDI-TOF, trypsin-digested proteins were mixed with
a saturated solution of ␣-cyano-4-hydroxycinnamic acid in 50%
acetonitrile containing 0.1% TFA and subjected to MALDI-
TOF analysis (Ettan MALDI-ToF Pro, Amersham Biosciences)
as described previously (29). Spectra were collected from 350
shots per spectrum over an m/z range of 600 –3000 and cali-
brated by a two-point internal calibration using trypsin auto-di-
gestion peaks (m/z 842.5099 and 2211.1046). A peak list was gen-
erated using the Ettan MALDI-TOF Pro Evaluation Module
(version 2.0.16). The threshold used for peak picking was as fol-
lows: 5,000 for minimum resolution of monoisotopic mass, 2.5 for
signal-to-noise ratio. The search program MASCOT, developed
by Matrix Science, was used for protein identification by peptide
mass fingerprinting. The following parameters were used for the
database search: trypsin as the cleaving enzyme, a maximum of
one missed cleavage, iodoacetamide (Cys) as a complete modifica-
tion, oxidation (Met) as a partial modification, monoisotopic
masses, and a mass tolerance of ⫾ 0.1 Da. Peptide mass fingerprint
acceptance criteria is probability scoring.
5⬘- and 3⬘-RACE Analysis of Os12g0240900—Because expressed
mRNA of Os12g0240900 had not been previously identified, we
used estimated ORF information in the rice genomic sequence
of the locus for RACE analysis. Extraction of total RNA from
UV-irradiated wild-type rice leaves (cv. Nipponbare) was con-
ducted using Sepasol-RNA I Super (Nakalai Tesque). and the
mRNA was then purified using the Absolutely mRNATM puri-
fication kit (Agilent) from the total RNA. cDNA synthesis from
the mRNA and the subsequent 5⬘- and 3⬘-RACE analyses were
performed using the SMARTerTM RACE cDNA amplification
kit (Clontech). Primers used in 5⬘- and 3⬘-RACE analyses are
described in supplemental Table S1. The 1,548-bp sequence of
the obtained cDNA of Os12g0240900 was deposited in the
DNA Data Bank of Japan (DDBJ) under accession number
AB692949.
Cloning, Expression, and Purification of Os04g0175900 and
Os12g0240900—Full-length ORFs of Os04g0175900 and
Os12g0240900 were amplified by RT-PCR using KOD-FX
Neo (TOYOBO, Tokyo, Japan) with cDNA prepared from
CuCl2-treated wild-type rice leaves (cv. Nipponbare) as tem-
plates. To construct a directional TOPO cloning transfer
vector, the 5⬘-CACC-3⬘ sequence was incorporated into the
PCR upstream primer at its 5⬘ ends. Then the PCR products
were inserted into the pENTR/D-TOPO vector (Invitrogen),
and the ligated products were transformed into TOP10 chem-
ically competent E. coli. The ORFs of Os04g0175900 and
Os12g0240900 inserted into pENTR/D-TOPO vector were
fused into pDEST15 (Invitrogen) using LR Clonase II enzyme
mix (Invitrogen). Finally, we obtained expression vectors of
N-terminal GST tag-fused OMTs, pDEST15-1759 and
pDEST15-2409, respectively.
The resulting plasmids were transformed into E. coli Rosetta
II (DE3) (Novagen). These strains were pre-cultured for 24 h at
37 °C in LB medium containing carbenicillin (100 ␮g/ml) and
chloramphenicol (34 ␮g/ml) and then cultured for 48 h at 20 °C
VOLUME 287 • NUMBER 23 • JUNE 1, 2012
 Purification and Identification of Rice Sakuranetin Synthase
in Overnight ExpressTM instant TB Medium (Novagen) con-
taining carbenicillin (100 ␮g/ml) and chloramphenicol (34
␮g/ml). The cells were collected by centrifugation, suspended
in buffer A, and disrupted by mild sonication on ice. After cen-
trifugation at 10,000 ⫻ g for 15 min, the supernatant was col-
lected and loaded to the AKTA FPLC (GE Healthcare)
equipped with a pre-equilibrated GSTrap HP column (bed vol-
ume 5 ml) (GE Healthcare). Buffer A was used as the pre-equil-
ibration buffer. Applied protein was washed with 25 ml of
buffer A with a flow rate of 2 ml/min. Next, 25 ml of buffer A
containing 10 mM reduced glutathione was applied for the elu-
tion. The purified recombinant proteins were ultrafiltered (10-
kDa cutoff) and finally resolved in 1 ml of buffer A, and that
solution was used for kinetic analysis.
MS/MS Analysis of Sakuranetin Produced by NOMT Enzymatic
Assays—To confirm that GST-2409 product was sakuranetin,
we compared product ion spectrum of the product and authen-
tic sakuranetin with LC-MS/MS system. MS/MS analysis was
performed with AB SCIEX TripleTOFTM 5600 System (AB
SCIEX) equipped with SHIMADZU Nexera UFLC system (Shi-
madzu, Kyoto, Japan). Four-min gradient elution with 10 –90%
acetonitrile containing 0.1% formic acid was performed with a
CAPCELL PAK C18 IF S2 column (Shiseido Co., Tokyo, Japan).
Ionization was performed with electrospray ionization and pre-
cursor ions (m/z ⫽ 100 –1,000) and product ions (m/z ⫽
50 –1,000) were scanned with positive ion mode. The retention
time of sakuranetin was 2.85 min. The product ion spectrum of
precursor ion (m/z ⫽ 287.1) at the retention time of sakurane-
tin was observed.
Chiral Analysis of Sakuranetin Produced in the NOMT Enzy-
matic Assays—For chiral analysis of sakuranetin, 70 min of iso-
cratic elution with 30% acetonitrile containing 0.1% acetic acid
(v/v/v) was performed with a Sumichiral OA-7500 column (250
mm long, 2 mm in diameter; Sumika Chemical Analysis Ser-
vice, Tokyo, Japan) and detected by using API-3000 LC-MS/MS
system as described above. The retention times of natural and
unnatural sakuranetin were 54 and 61 min, respectively.
Kinetic Analysis—The standard assay mixture without the
enzyme was incubated at 30 °C for 5 min. The reaction was
initiated by adding 1 pmol of the enzyme, and the reaction
mixture was incubated at 30 °C for 5 min. The reaction was
terminated by adding 5 ␮l of 1 M HCl. The resulting products
were extracted three times with 60 ␮l of ethyl acetate, evapo-
rated to dryness, and finally dissolved in 100 ␮l of phytoalexin
extraction solvent. The content of sakuranetin was quantified
using an API-3000 LC-MS/MS system. The kinetic parameters
were derived from a Hanes-Woolf plot. The effect of substrate
concentration on reaction velocity was examined at various
concentrations (30, 24, 18, 12, 6, and 0 ␮M) of racemic nar-
ingenin, unnatural naringenin being not a substrate for
OsNOMT. The concentration of natural naringenin was calcu-
lated as half of racemic naringenin.
Substrate Specificity Assay—The standard assay mixture
without AdoMet was incubated at 30 °C for 5 min. The reaction
was initiated by adding 45.9 pmol (1 ␮l) of S-[methyl-3H]
adenosyl-L-methionine (⬃444 GBq/mmol, 20.4 MBq/ml,
PerkinElmer Life Sciences), and the reaction mixture was incu-
bated at 30 °C for 10 min. The reaction was terminated by add-
JUNE 1, 2012 • VOLUME 287 • NUMBER 23
ing 5 ␮l of 1 M HCl. The resulting products were extracted with
150 ␮l of ethyl acetate, and 75 ␮l of the extract was applied to a
liquid scintillation counter.
Preparation and Inoculation of Fungal Conidia—The rice
blast fungus M. oryzae (race 001.0 P91-15B) was kindly sup-
plied by Dr. E. Minami (National Institute of Agrobiological
Sciences). The blast fungi were grown on oatmeal agar (60 g
of homogenized oatmeal and 12 g of agar per liter of water) in
Petri dishes incubated at 25 °C for 9 days. Conidial suspen-
sion was prepared as described previously (30) and then fil-
tered through two layers of Miracloth (Calbiochem) to
remove cell debris. Conidial suspension was subjected to an
inoculation test within 2 h after preparation. For inocula-
tion, the excised fourth leaf blades of rice plants at the 4.5-
leaf stage were sprayed with conidial suspension at a concen-
tration of 105 spores/ml. Then the inoculated leaf blades
were incubated for 120 h at 25 °C under light conditions, and
the samples were applied for gene expression analysis and
quantitative analysis of sakuranetin.
RESULTS
Characterization of oscomt1 Mutant—We obtained PFG-
2B-50240 (cv. Dongjin) as a tDNA insertion mutant in the
first exon of Os08g0157500 (TIGR ID, LOC_Os08g06100,
OsCOMT1) (Fig. 2A) from the Rice Functional Genomic
Express Database and selected the oscomt1 homozygote
mutant and wild-type rice from the F2 generation of seeds
obtained by genomic PCR (Fig. 2B). After confirmation of
impaired transcriptional expression of the OsCOMT1 gene
in leaves of the oscomt1 mutant by RT-PCR (Fig. 2C), we
examined the ability of the oscomt1 mutant leaves to produce
sakuranetin. Excised leaf blades of oscomt1 and wild-type
rice were treated with CuCl2, and the accumulated amounts
of sakuranetin in the leaf blades 72 h after treatment were
determined by LC-MS/MS analysis. As shown in Fig. 3A, the
accumulation of sakuranetin in oscomt1 was comparable
with that in the wild type. We also compared NOMT activity
and COMT activity in leaf blades of oscomt1 and the wild
type. Crude enzyme extracts derived from rice leaves of
oscomt1 and the wild type 48 h after CuCl2 treatment were
subjected to NOMT and COMT enzymatic assays in which
naringenin and caffeic acid were used as substrates, respec-
tively. To determine NOMT and COMT activity, the reac-
tion products of these assays, namely sakuranetin and ferulic
acid, were measured by LC-MS/MS. As shown in Fig. 3, B
and C, the specific NOMT activity of oscomt1 was compara-
ble with that of the wild type, whereas the specific COMT
activity of oscomt1 was severely suppressed compared with
that of the wild type. This indicates that UV-irradiated
oscomt1 leaf blades are suitable plant materials for purifica-
tion of OsNOMT.
Purification of OsNOMT from oscomt1 and MALDI-TOF-
TOF Analysis of Purified Protein—Because NOMT activity in
oscomt1, as already described, was comparable with that in the
wild type, we performed purification of OsNOMT from
oscomt1 leaves using a series of chromatographic steps. A crude
enzyme extract prepared from oscomt1 leaves 48 h after UV
treatment was purified through three steps of ammonium sul-
JOURNAL OF BIOLOGICAL CHEMISTRY 19319
Purification and Identification of Rice Sakuranetin Synthase

FIGURE 3. Accumulation of sakuranetin and NOMT and COMT enzymatic activity in elicited oscomt1 and wild-type rice leaves. A, accumulated sakurane-
tin in oscomt1 and wild-type rice leaves 48 h after CuCl2 treatment was determined by LC-MS/MS. B and C, specific NOMT (B) and COMT (C) activities of crude
enzyme preparations from oscomt1 and wild-type rice leaves 48 h after CuCl2 treatment. Amounts of sakuranetin and ferulic acid extracted from the enzymatic
reaction mixture were measured by LC-MS/MS, and the specific activities were determined. The data are means ⫾ S.D. of three replicates. An asterisk indicates
significant differences compared with wild-type rice in a t test at p ⬍ 0.05.

TABLE 1
Purification of OsNOMT from UV-irradiated oscomt1 rice leaves
Purification steps Total activity Total protein Specific activity Recovery Purification
picokatal mg picokatal/mg % -fold
Crude extract 104.0 695 0.150 100 1
Ammonium sulfate precipitation 43.6 288 0.152 42.0 1.01
Anion exchange (DEAE) 26.4 112.2 0.235 25.4 1.57
Affinity chromatography (adenosine-agarose) 5.8 0.096 60.629 5.6 405.35

fate precipitation, DEAE anion exchange chromatography, and
adenosine-agarose chromatography. The specific activity of
OsNOMT and the results of SDS-PAGE analysis at each step
are given in Table 1 and Fig. 4A, respectively. The specific activ-
ity of OsNOMT was dramatically concentrated by adenosine-
agarose chromatography, and a 40-kDa band was detected on
SDS-PAGE after adenosine-agarose chromatography. The
40-kDa band was excised from the gel and treated with trypsin,
and the products of tryptic digestion were analyzed by MALDI-
TOF/TOF MS. The obtained data were entered into the
MASCOT Database. As a result, two putative OMTs encoded
by loci Os04g0175900 (TIGR ID, LOC_Os04g09654) and
Os12g0240900 (TIGR ID, LOC_Os12g13800) were identified
as components of the 40-kDa band (Fig. 4, B and C). The mass
spectra of these OMTs are shown in supplemental Fig. S1.
Although proteins of 45 and 52 kDa were also detected as major
bands in the fraction after adenosine-agarose chromatography
(Fig. 4A), they were identified by MALDI-TOF MS as Rubisco-
related proteins (45-kDa protein, Rubisco large chain precursor
(AK058623, 40.2 kDa); 52-kDa protein, Rubisco large chain
(AK105600, 52.8 kDa)).
cDNA Cloning and Functional Identification of Os04g0175900 and
Os12g0240900—To investigate whether the Os04g0175900
and Os12g0240900 gene products have NOMT enzymatic
activity, their cDNAs were expressed as recombinant proteins
in E. coli. For preparation of the Os04g0175900 cDNA,
sequence information of AK104764 in the RAP-DB was used.
Because a full-length cDNA clone of Os12g0240900 had not
been previously isolated, 5⬘- and 3⬘-RACE analysis was per-
formed using predicted ORF information from rice genomic
sequence of this gene locus. Total RNA was extracted from
wild-type leaf blades 48 h after UV irradiation. mRNA was puri-
fied from the total RNA and subjected to cDNA synthesis fol-
lowed by 5⬘- and 3⬘-RACE PCR. Finally, the full-length cDNA
(1,548 bp) of Os12g0240900 was amplified by RT-PCR using
primer sets constructed based on the sequences of the 5⬘ and 3⬘
ends and deposited in the DNA Data Bank of Japan under
accession number AB692949.
The longest ORFs of Os04g0175900 (AK104764) and
Os12g0240900 (AB692949) were amplified by RT-PCR, cloned
using pENTR/D-TOPO vector (Invitrogen), incorporated into
the expression vector pDEST15 (Invitrogen), and overex-
pressed in E. coli as N-terminal GST-tagged proteins, desig-
nated as GST-1759 and GST-2409, respectively. The crude
extracts containing the recombinant proteins were subjected to
FPLC with a glutathione-Sepharose column (GSTrap HP,
Amersham Biosciences) to obtain the pure GST-1759 and
GST-2409. NOMT enzymatic activities of the purified recom-
binant proteins (Fig. 5A) were examined, and the Michaelis-
Menten kinetic parameters (Km and kcat values) were deter-
mined. As shown in Fig. 5B and supplemental Fig. S2,
sakuranetin was detected as a product of the enzymatic reac-
tion catalyzed by GST-2409 but not by GST-1759. As we used
racemic naringenin as a substrate, we confirmed by using chiral
column chromatography that sakuranetin produced by the
NOMT enzymatic assay with GST-2409 was a natural form
(supplemental Fig. S3). Km and kcat values of GST-2409 for nat-
ural naringenin determined by the Hanes-Woolf plot (Fig. 5C)
were 1.9 ⫾ 0.1 ␮M and 25 ⫾ 3/s, respectively. These results
indicate that the gene product of Os12g0240900, but not that of
Os04g0175900, can catalyze 7-O-methylation of naringenin.
We therefore designated Os12g0240900 as OsNOMT.
Substrate Specificity of GST-OsNOMT—It was reported that
F1-OMT and SaOMT-2 catalyze the methylation of several fla-
vonoids, including naringenin (20, 21). Therefore, we deter-
mined the substrate specificity of OsNOMT against several
types of phenolic compounds, including flavonoids and isofla-
vonoids. The relative activity of purified GST-OsNOMT was

19320 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 23 • JUNE 1, 2012

 Purification and Identification of Rice Sakuranetin Synthase

FIGURE 4. Identification of O-methyltransferase-like proteins by MALDI-TOF/TOF analysis. A, Coomassie-stained SDS-PAGE of purified enzyme prepara-
tions is shown. Lane 1, crude enzyme; lanes 2– 4, ammonium sulfate precipitation-, DEAE-, and adenosine-agarose affinity-purified enzymes; lane M, protein
marker. Black arrowhead indicates the 40-kDa proteins, which include OsNOMT. White arrowheads indicate the major components of Rubisco-related proteins.
B and C, amino acid sequences of O-methyltransferase-like proteins encoded by gene loci Os04g0175900 (B) and Os12g0240900 (C) obtained from the 40-kDa
band by MALDI-TOF/TOF analysis are shown. Bold italic characters indicate the fragments identified by MALDI-TOF/TOF analysis.

tested on the methylation of two flavanones (naringenin and
liquiritigenin), five flavones (apigenin, luteolin, kaempferol,
quercetin, and myricetin), an isoflavanone (daidzein), an isofla-
vone (biochanin A), caffeic acid, and sinapic acid. A mixture of
a substrate and the purified OsNOMT was incubated with 3H-la-
beled AdoMet for methylation quantification. The reaction prod-
uct was extracted with ethyl acetate, and its 3H radioactivity was
determined by liquid scintillation counter to evaluate the substrate
specificity of OsNOMT. As shown in Fig. 6, OsNOMT preferen-
tially methylated flavanones and flavones, showing the highest
methylation activity with racemic naringenin (100%), followed by
kaempferol (73%), apigenin (61%), luteolin (44%), racemic liquiriti-
genin (34%), and quercetin (30%). The methylation activity of
OsNOMT with myricetin, isoflavones, and other phenolics was
similar to that of the negative control.
Relationship between Expression of OsNOMT and Accumu-
lation of Sakuranetin in Rice Leaves after JA Treatment and M.
oryzae Infection—Accumulation of sakuranetin is induced by
treatment with elicitors, such as JA and CuCl2 in rice leaves,
and CuCl2 treatment induced JA prior to accumulation of

JUNE 1, 2012 • VOLUME 287 • NUMBER 23 JOURNAL OF BIOLOGICAL CHEMISTRY 19321

Purification and Identification of Rice Sakuranetin Synthase

FIGURE 5. NOMT enzymatic activity of GST-fused recombinant proteins. Crude enzyme preparations from E. coli expressing either GST-2409 or GST-1759
were purified by affinity chromatography with GSTrap HP column, and the purified preparations were applied to SDS-PAGE and NOMT enzymatic activity
assays. A, Coomassie-stained SDS-PAGE of affinity purified GST-1759 and GST-2409. (C, crude; P, purified). B, LC-MS/MS chromatograms of reaction products in
the NOMT enzymatic activity assay using either GST-2409 or GST-1759 and an authentic standard of sakuranetin. C, Hanes-Woolf plots for GST-2409 with
natural naringenin. The concentration of natural naringenin was calculated as the half of racemic naringenin. The data are means ⫾ S.E. of three replicates.

flavanone flavone
R1
OH OH

HO O HO O
naringenin a R2

kaempferol b R3
R O OH O
apigenin b
liquiritigenin: R=H apigenin: R1=R2=R3=H
luteolin c naringenin: R=OH luteolin: R1=OH, R2=R3=H
kaempferol: R1=R2=H, R3=OH
quercetin: R1=R3=OH, R2=H
liquiritigenin cd myricetin: R1=R2=R3=OH
quercetin d
isoflavanone isoflavone
myricetin e
HO O HO O
biochanin A e

daidzein e O OH O
OH OCH 3
caffeic acid e daidzein biochanin A

sinapic acid e
others
negative control e
HO COOH CH3O COOH
0 20 40 60 80 100 120
HO HO
relative methylation activity (%)
OCH3
caffeic acid sinapic acid

FIGURE 6. Relative methylation activities of OsNOMT against flavonoids and other phenolic compounds. Purified GST-OsNOMT was incubated with 0.3
mM of phenolic substrates and a trace amount of S-[methyl-3H]adenosyl-L-methionine. The reaction was terminated by adding 1 M HCl. The reaction products
were extracted with ethyl acetate and the incorporated methyl-3H was measured. The data are means ⫾ S.E. of three replicates. Different characters indicate
significant differences in a Tukey’s test (p ⬍ 0.05) among the activities against the substrates.

sakuranetin (17). Therefore, we performed time course ana-
lyses of expression levels of OsNOMT and accumulation of
sakuranetin to further support the involvement of OsNOMT
in sakuranetin production in rice leaves after JA treatment.
For JA treatment, rice leaf disks were floated on 100 ␮M JA.
Then the expression levels of OsNOMT in the leaf disks were
determined by qRT-PCR, and the endogenous levels of
sakuranetin were quantitated by LC-MS/MS. The mRNA
level of OsNOMT was transiently induced at 6 h and then
gradually decreased until 72 h after JA treatment (Fig. 7A).
Accumulation of sakuranetin started at 6 h and continuously
increased until at least 72 h after JA treatment, when the
accumulation level of sakuranetin reached ⬃4 ␮g/g FW (Fig.
7B).

19322 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 23 • JUNE 1, 2012

 Purification and Identification of Rice Sakuranetin Synthase
FIGURE 7. Time course analysis of OsNOMT mRNA and accumulation of
sakuranetin and naringenin in JA-treated wild-type rice leaves. A, rela-
tive expression levels of OsNOMT in rice leaves from 0 to 72 h after treat-
ment with 100 ␮M JA. The mRNA levels were determined using qRT-PCR.
Each value was normalized to the OsUBQ mRNA level. The data are
means ⫾ S.D. of three experiments. B and C, accumulated amounts of
sakuranetin (B) and naringenin (C) from 0 to 72 h after treatment with 100
␮M JA were quantified using LC-MS/MS. The data are means ⫾ S.E. of three
experiments.
In addition, we found that accumulation of naringenin, a
direct precursor of sakuranetin, was transiently induced
slightly prior to accumulation of sakuranetin, peaking at ⬃0.5
␮g/g FW 48 h after JA treatment (Fig. 7C). Thus, it was indi-
cated that when rice produces sakuranetin, both the expression
of the OsNOMT gene and the supply of the substrate nar-
ingenin are induced.
Besides, accumulation of sakuranetin is also induced by M.
oryzae infection. We confirmed up-regulation of OsNOMT
expression and increase in accumulation of both naringenin
and sakuranetin in rice leaves at 5 days post-inoculation with
M. oryzae spores (supplemental Fig. S4).
JUNE 1, 2012 • VOLUME 287 • NUMBER 23
DISCUSSION
Here, we present details of the purification of the rice narin-
genin 7-O-methyltransferase OsNOMT from rice oscomt1
mutant leaves and its detailed characterization. Although a
putative OsNOMT was previously partially purified from UV-
irradiated wild-type rice leaves, as described above, a major
protein in the purified fraction showed high homology to maize
COMT, and its recombinant protein expressed in E. coli
showed COMT, but not NOMT, activity and was named
OsCOMT1 (26).
Our study indicated that sakuranetin accumulation and
NOMT enzymatic activity were similarly induced after elicita-
tion in the oscomt1 mutant and wild-type rice leaves. This
strongly suggests that OsCOMT1 is not involved in sakuranetin
production in rice. A possible reason why OsNOMT was not
detected from the purified fraction with NOMT enzymatic
activity from crude extracts of UV-treated wild-type rice leaves
in the previous study (17) may be that the more abundantly
generated OsCOMT1 exhibited similar behavior to OsNOMT
in a series of purification steps and masked the presence of
OsNOMT. In this study, purification of OsNOMT was success-
fully performed without masking by OsCOMT1 by using elic-
ited oscomt1 leaves. Among the three purification steps of
OsNOMT, adenosine-agarose chromatography is most effec-
tive, leading to a more than 400-fold enrichment of the enzyme.
This chromatography is known to be very effective in purifying
AdoMet-dependent methyltransferases (31). After this purifi-
cation step, a 40-kDa band on SDS-PAGE was detected. It was
shown that molecular masses of type 1 OMTs, whose substrates
are caffeic acid, flavonoids, coumarin, and alkaloids, are about
38 – 43 kDa (32), and OsNOMT and OsCOMT1 were sug-
gested to have molecular masses of around 41 kDa (22). The
40-kDa band was excised from the gel and subjected to
MALDI-TOF/TOF MS analysis after treatment with trypsin,
resulting in identification of the two OMTs as candidate pro-
teins for OsNOMT. Preparation of GST-tagged gene products
of Os04g0175900 (AK104764) and Os12g0240900 (AB692949)
in E. coli followed by in vitro NOMT enzymatic assays clearly
demonstrated that Os12g0240900 encodes OsNOMT.
Kinetic analysis of recombinant GST-OsNOMT revealed
that the Km of GST-OsNOMT for naringenin was ⬃1.9 ␮M.
Conversely, the endogenous level of naringenin accumulation
was ⬃1.8 ␮M in rice leaves 48 h after JA treatment (Fig. 7C).
Taken together, the Km value was quite reasonable in under-
standing the function of OsNOMT in sakuranetin production
in rice leaves.
Substrate specificity of OsNOMT was also determined in
this study as described by Christensen et al. (20), and the results
revealed that GST-OsNOMT showed higher methylation
activity on naringenin than the flavanone liquiritigenin and fla-
vones and no methylation activity on other phenolics, including
isoflavonoids (Fig. 6). In the case of F1-OMT in barley, the
methylation activity on apigenin was three times higher than
that on naringenin (20). In contrast, SaOMT-2 has broad sub-
strate specificity and can catalyze the methylation of isofla-
vonoids as well as naringenin (21). Sakuranetin has not been
identified in either barley or S. avermitilis. These results indi-
JOURNAL OF BIOLOGICAL CHEMISTRY 19323
Purification and Identification of Rice Sakuranetin Synthase
cate that the actual biological functions of F1-OMT and
SaOMT-2 are still unknown.
Time course analyses of OsNOMT expression and of accu-
mulation levels of sakuranetin and its direct precursor narin-
genin in rice leaves treated with JA indicate that OsNOMT
expression was induced transiently prior to accumulation of
sakuranetin, and accumulation of naringenin was also tran-
siently induced slightly prior to that of sakuranetin (Fig. 7C).
Up-regulation of OsNOMT was confirmed in the pathogen M.
oryzae-infected rice leaves in which accumulation of sakurane-
tin and its precursor naringenin was increased (supplemental
Fig. S3). These results further support that OsNOMT functions
as a sakuranetin synthase and is involved in defense responses
through elicitor-induced production of sakuranetin in rice. The
success of identification of OsNOMT as a sakuranetin synthase
in this study will enable enhancement of pathogen resistance
through regulation of the endogenous content of sakuranetin in
rice. In addition, as described in Introduction, sakuranetin is a
useful compound showing various pharmaceutical activities
(12–16). Because the fermentative production of naringenin by
microorganisms carrying an artificially assembled phenylpro-
panoid pathway has been established (33), our success in clon-
ing OsNOMT will also enable the production of a large amount
of sakuranetin by microorganisms for medical research.
Acknowledgments—We thank Dr. H. Nagasawa, Dr. S. Nagata, and
M. Kuroiwa (Graduate School of Agricultural and Life Sciences, The
University of Tokyo) for their support with purification and identifi-
cation of OsNOMT. We thank Dr. T. Kuzuyama and T. Ozaki (Bio-
technology Research Center, The University of Tokyo) for their support
with product ion analysis with LS-MS/MS of sakuranetin. We thank
Dr. E. Minami (National Institute of Agrobiological Sciences) for
sharing the culture of M. oryzae used in this study. We thank Dr.
Gynheung An (Institution and Pohang University of Science and
Technology) for providing the oscomt1 mutant.
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